arf2 coordinates with plethoras and pins to orchestrate ... · arf2 coordinates with plethoras and...

ARF2 coordinates with PLETHORAs and PINs toorchestrate ABA-mediated root meristem activityin ArabidopsisFA

Sujittra Promchuea1, Yujuan Zhu1, Zhizhong Chen1, Jing Zhang1 and Zhizhong Gong1,2*

1. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China2. National Center for Plant Gene Research, Beijing 100193, China*Correspondence: Zhizhong Gong ([email protected])doi: 10.1111/jipb.12506

Abstract Multiple hormones, including abscisic acid(ABA) and auxin, regulate cell division and differentiationof Arabidopsis root meristems. AUXIN RESPONSE FACTOR2 (ARF2) functions as a negative regulator of ABAresponses, as seed germination and primary root growthof arf2mutants are hypersensitive to ABA. In this study, wefound that ABA treatment reduced the expression levels ofthe PIN-FORMEDs (PIN) auxin efflux carriers, PIN1, PIN3,PIN4, and PIN7, to a greater extent in the root meristemsof arf2-101 mutant than in the wild type. Also, arf2-101 pin1and arf2-101 pin4 double mutants show less ABA-inducedinhibition of root meristem activity than the arf2-101mutants. Furthermore, ARF2 positively mediates thetranscripts of transcription factor PLETHORA 1 (PLT1)gene but negatively mediates PLT2 at protein level in

root meristems. Using a dexamethasone (DEX)-inducibletransgenic line, Pro35S:PLT2-GR, we showed that PLT2greatly promotes cell division and completely inhibits celldifferentiation in root meristems of the arf2-101 mutantonce PLT2 is induced by DEX, which can be partiallyreversed by ABA treatment, suggesting that ABA regulatesroot meristem activity in both ARF2-dependent andindependent pathways. Our results uncover a complexregulatory architecture in which ARF2 coordinates withPLTs and PINs to orchestrate ABA-mediated regulation ofroot meristem activity in Arabidopsis.

Edited by: Alice Y. Cheung, University of Massachusetts, USAReceived Oct. 7, 2016; Accepted Nov. 1, 2016; Online on Nov. 7,2016

FA: Free Access, paid by JIPB

INTRODUCTION

Root growth is dynamically regulated by a network ofinteracting hormone signals that coordinate celldivision and differentiation to maintain the root apicalmeristem (RAM) (Lee et al. 2013). Water stress inducesthe accumulation of ABA, which can promote primaryroot growth at low concentrations and inhibit primaryroot growth at high concentrations (Zhang et al.2010). Some DNA replication-related mutants havesmall RAMs and are hypersensitive to ABA, suggestingthat ABA inhibits cell division through DNA replication-related proteins (Yin et al. 2009; Yao et al. 2013).Previous studies indicated that ABA signaling couldpromote ethylene production via phosphorylation

of the C-termini of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS), and stabilizationof ACSs by ABA-activated calcium-dependent proteinkinase 4 (CPK4) and CPK11 (Luo et al. 2014). Ethylenesignaling upregulates WEAK ETHYLENE INSENSITIVE2(WEI2)/ANTHRANILATE SYNTHASE alpha1 and WEI7//ANTHRANILATE SYNTHASE beta1 to synthesize auxinand inhibit primary root growth (Stepanova et al.2005; Mao et al. 2016). ABA also promotes theproduction of reactive oxygen species in mitochon-dria, reduces auxin accumulation, and results in theinhibition of primary root growth (He et al. 2012; Yanget al. 2014).

Auxin plays important roles in RAM establishmentand maintenance (Overvoorde et al. 2010). The PIN-

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FORMEDs (PINs) polar auxin efflux transporters arecritical for producing a proper auxin gradient in RAMs(Blilou et al. 2005; Petrasek and Friml 2009). In theprimary root, basally localized PIN1, PIN3, and PIN7 inthe stele mediate auxin flow towards the tip, whichleads to high concentrations of auxin in the stem cellniche. PIN4, PIN3, and PIN7 expressed in the columellacells distribute auxin to the root cap and the epidermis,where the apically localized PIN2 mediates the upwardflow of auxin to the elongation zone (Friml et al. 2002;Friml et al. 2003; Friml et al. 2004; Blilou et al. 2005;Petrasek and Friml 2009). In RAMs, key transcriptionfactors such as WUSCHEL-RELATED HOMEOBOX 5,PLETHORA (PLT), SHORT ROOT, and SCARECROW,which respond to intercellular signaling, ultimatelyregulate cell division and differentiation (DiLaurenzioet al. 1996; Helariutta et al. 2000; Sabatini et al. 2003;Aida et al. 2004; Blilou et al. 2005; Sarkar et al. 2007).Recent studies indicated that the levels of PLT proteinsplay crucial roles in determining cell fate in the RAM(Galinha et al. 2007; Mahonen et al. 2014). A PIN-mediated auxin gradient underlies the PLT gradientsand the PLT gradients in turn affect PIN levels (Frimlet al. 2003; Blilou et al. 2005; Galinha et al. 2007;Mahonen et al. 2014). However, prolonged high auxinlevels promote a narrow domain of PLT transcriptionfrom which a PLT gradient is produced to define thedevelopmental zones (Mahonen et al. 2014). Theexpression of PLTs is modulated by different factors,including the auxin response factors MONOPTEROS(MP), NON-PHOTO-TROPIC HYPOCOTYL4 (NPH4) (Aidaet al. 2004), MYC2 (Chen et al. 2011b), and RopGEF7(Chen et al. 2011a), and receptor-like protein kinasesRGFR1/2/3 (Shinohara et al. 2016), and a SWI/SNFchromatin remodeling ATPase BRAHMA (Yang et al.2015). High PLT levels in root stem cell niches promotestem cell identity andmaintenance,moderate PLT levelspromote stem cell division, and low PLT levels arerequired for cell differentiation (Galinha et al. 2007;Mahonen et al. 2014).

Previous studies indicated that ARF2 has multipleroles in regulating plant development and responses todifferent hormones (Li et al. 2004; Okushima et al. 2005;Schruff et al. 2006; Vert et al. 2008; Wang et al. 2011).ARF2 inhibits the expression of the homeodomain geneHB33, which mediates the ABA response in Arabidopsis(Wang et al. 2011). The root growth of the arf2mutantsis hypersensitive to ABA, but this sensitivity can be

rescued by the addition of the auxin influx inhibitor1-naphthoxyacetic acid (Wang et al. 2011). These resultssuggest that ABA inhibition of root growth in the arf2mutant involves auxin signaling (Wang et al. 2011). Inthis study, we further explored themechanism for ARF2regulation of root growth in ABA signaling. We foundthat the arf2 mutant changes the expression of PINs,PLT1, and PLT2. Interestingly, dexamethasone (DEX)-induced PLT2-GR in the Pro35S:PLT2-GR transgenic linetriggers increased cell division but completely blockscell differentiation in the RAM of the arf2 mutant, andthis could be partially inhibited by ABA treatment. Ourresults thus uncover an important interplay amongARF2, PINs, and PLT2 in ABA-mediated root meristemactivity.

RESULTS

ARF2 regulates RAM activity and cell elongation inABA signalingA previous study indicated that ARF2 regulates rootgrowth through ABA signaling (Wang et al. 2011). Thearf2-101 mutant showed enhanced ABA sensitivity inseed germination and primary root growth (Wang et al.2011). Under normal conditions, the root meristem zoneand elongation zone were slightly smaller in the arf2-101mutant than in the wild type (WT) (Figure 1A�C). UnderABA treatment, the length of the meristem zone (MZ)and cell number in the MZ were significantly reduced inthe arf2-101 mutant, but the average cell length in theMZ did not change much when compared with the WT(Figure 1A�C). The length of the elongation zone (EZ)and cell length in the EZ, but not cell number, were allsignificantly reduced in the arf2-101 mutant after ABAtreatment, compared with the WT (Figure 1A, B, D).These results indicate that ARF2 positively regulatesRAM activity in ABA signaling.

ARF2 alters the accumulation of PIN proteins in theRAM after ABA treatment

A previous study showed that the expression of PIN3and PIN7 decreased in the arf2-101 mutant under ABAtreatment, compared with the WT (Wang et al. 2011).In this study, we examined the expression of PINsby crossing the arf2-101 mutant with constructsencoding green fluorescent protein (GFP) fusions tothe PIN proteins, expressed under the control of their

ARF2 mediates PLTs and PINs in ABA signaling 31

www.jipb.net January 2017 | Volume 59 | Issue 1 | 30–43

native promoters. We found that the levels of PIN1-GFP, PIN3-GFP, and PIN7-GFP in the root tips of thearf2-101 plants with the reporter constructs werehigher than those in the WT grown on MS medium,and the expression of PIN4-GFP significantly increasedin the ProPIN4:PIN4-GFP arf2-101 transgenic plants

relative to the WT (Figure 2A�H). ABA treatmentdecreased the signals for all PINs in the arf2-101transgenic plants and in the WT, but the GFP signalsdecreased more in arf2-101 than in WT (Figure 2A�H).We also noted that after ABA treatment, the PIN4-GFPsignal was hardly detected in the WT, but could be

Figure 1. Abscisic acid (ABA) treatment inhibits the meristem and elongation zones in Col-0 and the arf2-101mutant(A) Comparison of root growth in the wild type (WT) and arf2-101 mutants. Four-day-old seedlings grown on MSmedium were transferred onto MS medium (control) or MS medium with 30mM ABA for 3 d. The meristem zone(MZ): red line; elongation zone (EZ): white line; and differentiation zone (DZ): green line. Scale bars¼ 100mm. Eachimage was made by joining several photographs of the same root. (B) A closer look at the root meristemphenotypes of Col-0 and the arf2-101 mutant seedlings in (A). The meristem zone is marked with a red line.Bars¼ 50mm. (C) The root meristem zone (MZ) length, cell number, and average cell length of theWT and the arf2-101mutant in (A). (D) The root elongation zone (EZ) length, cell number and average cell length of the WT and thearf2-101 mutant (A). Three independent experiments were conducted with similar results in (B) and (C). Eachexperiment was conducted with 12-25 roots from three plates. Values are means� SD. Means with different lettersare significantly different at P< 0.01.

32 Promchuea et al.


clearly observed in the ProPIN4:PIN4-GFP arf2-101transgenic plants. These results indicate that ARF2mediates the expression of PINs in the RAM throughABA signaling.

PIN1 and PIN4 negatively mediate RAM activity of thearf2-101 mutant in ABA signalingWhen we introduced the pin1-1, pin4-3, pin3-4, andpin7-2 mutations into the arf2-101 mutant, we foundthat pin1-1 and pin4-3 could partially suppress the ABA-sensitive phenotype of the arf2-101 mutant, but pin3-4and pin7-2 could not (Figures S1�S4), and pin1-1conferred slightly more sensitivity to ABA than the

WT. The arf2-101 pin1-1 double mutant had a smallerRAM than the arf2-101 or pin1-1 single mutants undernormal growth conditions (Figure 3A�C). Under a 10or 30mM ABA treatment, the arf2-101 pin1-1 doublemutant had a larger RAM and longer cells in theElongation zone (EZ) than the arf2-101 mutant(Figure 3A�C). PIN4 is associated with root meristemactivity and patterning (Friml et al. 2002; Garay-Arroyoet al. 2013). In both the control and in the ABAtreatment, the arf2-101 pin4-3 double mutant had alarger meristem than the arf2-101 mutant (Figure 4A).The arf2-101 pin4-3 double mutant had more cellnumber and longer zone length than afr2-101 in

Figure 2. The Expression of PIN1-, PIN3-, PIN4-, and PIN7-GFP in primary root tips of the arf2-101 mutantFluorescence images and the relative fluorescence of ProPIN1:PIN1-GFP (A and B), ProPIN3:PIN3-GFP (C and D),ProPIN4:PIN4-GFP (E and F), and ProPIN7:PIN7-GFP (G and H) in the root tips of the wild type (Col-0) and the arf2-101mutant. Four-day-old seedlings were transferred onto MS medium or MS medium supplemented with 30mMabscisic acid (ABA) for 2 d. Three experiments were conducted with similar results. Each experiment was conductedwith 8-15 roots from three plates. Values are means� SD. P< 0.01. Means with different letters are significantlydifferent at P< 0.01. Scale bar¼ 50mm.



meristem zone (Figure 4B), and longer cell length in EZ(Figure 4C) under ABA treatment. These resultssuggest that PIN1 and PIN4 negatively regulate RAMactivity of the arf2-101 mutant under ABA signaling.

ARF2 affects the expression of PLT1 and PLT2 in rootmeristemsThe PLETHORA genes have crucial functions in rootpatterning and auxin readout mechanisms, and the

transcriptional gradients of PLT1 and PLT2 stronglycorrelate with the auxin gradients in the RAM(Aida et al. 2004; Galinha et al. 2007). Transcrip-tion of PLT genes is stimulated by auxin andis dependent on ARF transcription factors suchas MONOPTEROS/ARF5 and NONPHOTOTROPIC HY-POCOTYL 4/ARF7 (Aida et al. 2004). To test whetherARF2 has any effect on the expression of PLT1and PLT2 in root apical meristems, we crossed the

Figure 3. The pin1 mutation reduces ABA-mediated inhibition of RAM activity in the arf2-101 mutant(A) The primary root meristem of the Col-0 and En ecotypes, the arf2-101, pin1-1 (En), and arf2-101 pin1-1 mutantseedlings on MS medium with and without ABA. Five-day-old seedlings were transferred to MS medium or MSsupplementedwith different concentrations of ABA for 5 d before taking images. Themeristem zone ismarkedwitha red line. The epidermal cell shapes are outlined in red in the 30mM ABA treatment for comparison of cell size.Bars¼ 50mm. (B) The root meristem zone (MZ) cell number, zone length and average cell length of each ecotype(A). (C) The root elongation zone (EZ) cell number, zone length and average cell length of each ecotype in (A). Threeindependent experiments were conducted with similar results in (B) and (C). Each experiment was conducted with12–25 roots from three plates. Values are means� SD. Means with different letters are significantly different atP< 0.01.

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arf2-101 mutant with the transgenic plants carryingProPLT1:PLT1-YFP, ProPLT2:PLT2-YFP, ProPLT1:CFP orProPLT2:CFP. The expression of ProPLT1:PLT1-YFPand ProPLT1:CFP was lower in the arf2-101 trans-genic plants than in the corresponding transgenicWT (Figure 5A, C, E, G). ABA treatment reduced theexpression of ProPLT1:PLT1-YFP and ProPLT1:CFP inboth the arf2-101 transgenic plants and in thecorresponding transgenic WT (Figure 5A, B, E, F).These results suggest that ARF2 positively regulatesthe expression of PLT1.

The expression of PLT2-YFP was slightly reduced inthe RAM of the ProPLT2:PLT2-YFP arf2-101 transgenic

plants compared with the ProPLT2:PLT2-YFP/Col-0root under normal conditions (Figure 5C, D). ABAtreatment at different concentrations reduced theexpression of ProPLT2:PLT2-YFP in the WT root, but hadno effect on its accumulation of PLT2-YFP in theProPLT2:PLT2-YFP arf2-101 plants (Figure 5C, D). How-ever, ProPLT2:CFP expression was lower in theProPLT2:CFP arf2-101 plants than in the ProPLT2:CFP/Col-0 plants under normal conditions, and wasreduced by ABA treatment to a similar level in bothplants (Figure 5G, H). These results suggest that ARF2negatively regulates PLT2 at the post-transcriptionallevel in ABA signaling.

Figure 4. The pin4mutation reduces abscisic acid (ABA)-mediated inhibition of RAM activity in the arf2-101mutant(A) The root meristem phenotype of Col-0 and the arf2-101, pin4-3 and arf2-101 pin4-3 mutants after 5-day-oldseedlings were transferred onto MS medium or MS medium supplemented with 30mM ABA for 5 d. The meristemzone is marked with a red line. Bars¼ 50mm. (B) The root meristem zone (MZ) cell number, zone length, andaverage cell length of each ecotype in (A). (C) The root elongation zone (EZ) cell number, zone length and averagecell length of each ecotype in (A). Three independent experiments were conducted with similar results in (B) and(C). Each experiment was conducted with 12�25 roots from three plates. Values are means� SD. Means withdifferent letters are significantly different at P< 0.01.



Overexpression of PLT2 promotes cell division andprevents cell differentiation in the arf2-101 mutantAlthough PLTs are functionally redundant and theiractivities in regulating the cell fate of the RAM arelargely additive and dosage dependent, the differentmodes by which AFR2modulates PLT1 and PLT2 suggestthat PLT1 and PLT2 may have some independent roles inmediating root growth in ABA signaling. As PLT2 is likelyregulated by ARF2 at the post-transcriptional level, tostudy the effect of PLT2 on root growth, we introduceda DEX-inducible PLT2-GR into the arf2-101 mutant bycrossing a Pro35S:PLT2-Glucocorticoid Receptor (Pro35S:PLT2-GR) transgenic plant with the arf2-101 mutant. Weobserved root meristem patterns directly after seedgermination and after the 4-day-old seedlings weretransferred onto DEX containing medium for 3 d. Theroot meristem cell number of the Pro35S:PLT2-GR/Col-0plants increased from 38, with no DEX induction, to 43after DEX treatment (Figure 6A�C). Surprisingly, theroot meristem cell number of the Pro35S:PLT2-GR/arf2-

101 transgenic plants increased from 43, with no DEXinduction, to 173 after DEX treatment (Figure 6A�C).Similarly, the root meristem cell number of the Pro35S:PLT2-GR/Col-0 plants increased from 28, with no DEXinduction, to 35 after DEX treatment, and from 31, withno DEX induction, to 157 after DEX treatment in thePro35S:PLT2-GR/arf2-101 transgenic plants after seedgermination for 6 d (Figure S5). The root meristemgrowth patterns indicated that once PLT2 is inducedby DEX, it promotes cell division and at the sametime completely blocks cell differentiation in the arf2-101 mutant. These results suggest that ARF2 hasan antagonistic effect on PLT2 in modulating celldivision and differentiation and PLT2 acts downstreamof ARF2.

We further examined the effect of ABA on rootmeristem cell number in these DEX-inducible lines.According to the sensitivity of seed germination androot growth to ABA, we used two concentrations ofABA: 30mM for root growth and 0.3mM for seed

Figure 5. ARF2 affects the expression of PLT1 and PLT2(A, B, E, F) The expression of ProPLT1:PLT1-YFP (A) and ProPLT1:CFP (E) in the arf2-101mutant and the WT. Four-day-old seedlings were transferred onto MS medium or MS medium supplemented with 30mM ABA for 2 d. Scalebar¼ 50mm.The relative fluorescence of ProPLT1:PLT1-YFP (B) in (A) and ProPLT1:CFP (F) in (E). Each experiment wasconductedwith 8-15 roots. Values aremeans� SD.Meanswith different letters are significantly different at P< 0.01.(C, D, G, H) The expression of ProPLT2:PLT2-YFP (C) and ProPLT2:CFP (G). Four-day-old seedlings were treated with 0,10, 20, and 30mM ABA for 2 d. Scale bar¼ 50mm. The relative fluorescence of ProPLT2:PLT2-YFP (D) in (C) andProPLT2:CFP (H) in (G). Each experiment was conducted with 8-15 roots. Values are means� SD. Means withdifferent letters are significantly different at P< 0.01.

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germination. Without DEX induction, ABA treatmentdecreased the root meristem cell number of theseedlings from 38 to 16 in the Pro35S:PLT2-GR/Col-0plants and from 43 to 17 in the Pro35S:PLT2-GR/arf2-101transgenic plants, and from 42 to 22 in the Pro35S:PLT2-GR/Col-0 plants and from 173 to 47 in the Pro35S:PLT2-GR/

arf2-101 transgenic plants with DEX induction(Figure 6A�C). Without DEX induction, ABA treatmentdecreased the root meristem cell number from 28to 18 in the Pro35S:PLT2-GR/Col-0 plants and from 31to 23 in the Pro35S:PLT2-GR/arf2-101 transgenic plantsafter seed germination for 6 d (Figure S5). With

Figure 6. Overexpression of PLT2 in the arf2-101 mutant promotes root meristem cell division and inhibits celldifferentiation(A) Seedling growth phenotypes after abscisic acid (ABA) and dexamethasone (DEX) treatment. Four-day-oldseedlings were transferred to MS medium or MS medium supplemented with 2mMDEX, 30mMABA or 30mMABAplus 2mM DEX for 3 d. (B) Comparison of root meristems of Pro35S:PLT2-GR/Col-0 and Pro35S:PLT2-GR/arf2-101seedlings in (A). The meristem zone is marked with a red line. Scale bars¼ 50mm. (C) Statistical analyses of rootmeristem number in (B). Three experiments were conducted with similar results. Each experiment was conductedwith 12-25 roots. Values are means� SD.



DEX induction, ABA treatment decreased the rootmeristem cell number from 35 to 22 in the Pro35S:PLT2-GR/Col-0 plants and from 157 to 49 in the Pro35S:PLT2-GR/arf2-101 transgenic plants after seed germinationfor 6 d (Figure S5). These results suggest that the highinduction of cell division by DEX-induced PLT2-GR in thearf2-101 mutant could be partially inhibited by ABAtreatment, and ABA inhibits cell division in both ARF2-dependent and �independent manners.

ARF2 mediates meristem cell division in parallel withPLT1, but not with PLT2ARF2, PLT1, and PLT2 are negative regulators in ABA-mediated root growth (Wang et al. 2011; Yang et al.2014). A previous study indicated that both the plt1 andplt2 single mutants display a slight but significantreduction in the growth rate and in the number ofmeristematic cells (Aida et al. 2004). To examine thegenetic relationship between ARF2 and PLT1/2 inregulating meristem cell division, we generated doublemutants by crossing arf2-101 with plt1-4 and plt2-2 andobservedmeristem size. The root meristems of the arf2-101 plt1-4 doublemutantswere smaller than those of thearf2-101 and plt1-4 single mutants on the controlmedium, suggesting that ARF2 and PLT1 have additivefunctions inmediating rootmeristem size (Figure 7A, B).The root meristem size of the arf2-101plt2-2 doublemutant was similar to that of the plt2-2 mutant andslightly smaller than that of the arf2-101 mutant. UnderABA treatment, the meristem size of the arf2-101 plt1-4double mutant was much smaller than either the arf2-101 or plt1-4 single mutants, further indicating that ARF2and PLT1 act in a parallel manner to mediate rootmeristem size under ABA signaling. In contrast, the rootmeristem size of the arf2-101 plt2-2 double mutant underABA treatment was similar to that of the arf2-101mutant(Figure 7A, B), suggesting that a mutation in PLT2 doesnot further enhance the ABA sensitivity of the arf2-101mutant in root growth. These results suggest that ARF2acts synergistically with PLT1, and ARF2 and PLT2 do nothave an additive effect on ABA-mediated root meristemactivity.

DISCUSSION

Plant root growth is regulated by different hormonesthat have complex crosstalk with auxin. The auxin

gradient in the root tip plays an important role inregulating root meristem division, differentiation, androot elongation (Aida et al. 2004; Galinha et al. 2007;Mahonen et al. 2014). Five auxin efflux transporter PINgenes (PIN1, 2, 3, 4 and 7) are expressed in the roots ofArabidopsis. The PIN-dependent auxin distributionnetwork involves redundancy and auxin-mediatedcross-regulation of PIN expression and PIN targeting(Blilou et al. 2005; Vieten et al. 2005). ABA treatmentinhibits both RAM activity and cell elongation in theelongation zone. Interestingly, we found that theexpression of PIN1, PIN2, PIN3, PIN4, and PIN7 geneswas higher in the arf2-101 mutant than in the WT undernormal conditions. ABA treatment reduced theirexpression to a greater extent in the arf2-101 mutantthan in the WT. Both the pin1 and pin4 mutationsincreased RAM activity in the arf2-101mutant under ABAtreatment, suggesting that PIN1 and PIN4 negativelymediate RAM activity in the arf2-101 mutant under ABAsignaling. In contrast, the pin3 and pin7 mutations didnot exert an effect on RAM activity in the arf2-101mutant. PIN1 is mainly expressed in the vascular cylindertissues, whereas PIN4 expression is localized in thequiescent center and surrounding cells of the rootmeristem (Friml et al. 2004; Blilou et al. 2005; Petrasekand Friml 2009). A recent study indicated that auxinconcentrations in tissues is regulated by nonpolar AUX1/LIKE-AUXIN influx carriers, whereas the direction ofauxin transport within the tissue is controlled by thepolar transporter PINs (Band et al. 2014). Under ABAtreatment, ProIAA2:GUS (an endogenous auxin marker)staining is lower in the arf2-101 mutant than in the WT,suggesting that the arf2-101 mutant roots mightaccumulate less auxin (Wang et al. 2011). However,the aux1 mutation is able to completely reverse thehyper-sensitivity to ABA of the arf2-101 mutant (Wanget al. 2011), while the aux1 mutant accumulates lessauxin in the root tips than the wild type (Swarup et al.2001). We also found that root growth in the pin1mutant is more sensitive to ABA than in theWT becausethe pin1 mutation did not enhance, but partiallyalleviated the ABA sensitivity of the arf2-101 mutant.These results suggest that the ABA sensitivity in theroots of the arf2 mutant is most likely due to auxindistribution and polar transporting.

Besides the PIN proteins, different transcriptionfactors, such as PLT1, PLT2, PLT3, and PLT4, actdownstream of auxin signaling in roots to regulate

38 Promchuea et al.


stem cell fate and cell division (Aida et al. 2004;Galinha et al. 2007; Mahonen et al. 2014). In this study,we found that the expression of PLT1 at both thetranscriptional and translational level was lower in thearf2-101 mutant than in the WT with and without ABAtreatment, suggesting that PLT1 expression is affectedby ARF2. However, our results demonstrated that theplt1 and arf2 mutations have an additive influence onthe ABA-inhibited RAM activity, suggesting that ARF2and PLT1 do not work in the same pathway. Thereduced expression of PLT1 in the arf2-101 mutantmight be caused by the indirect influence of ARF2.

Interestingly, the expression of PLT2 at both thetranscriptional and translational level is slightly lowerin the arf2-101 mutant than in the WT under normalgrowth conditions. ABA treatment reduced thetranscriptional level of PLT2 to a similar level in thearf2-101 mutant and in the wild type and consistentlyreduced PLT2 in the wild type, but did not change PLT2protein in the arf2-101 mutant. Given that PLT2transcripts are significantly reduced by ABA, theunchanged PLT2 protein level suggests that PLT2 ismore stable in the arf2-101 mutant than in the WTunder ABA treatment. As ARF2 does not directly

Figure 7. The plt1-4 mutation, but not the plt2-2 mutation, enhances the ABA-mediated inhibition of the rootmeristem in the arf2-101 mutant(A) The root meristem phenotypes of the Col-0 and Ws ecotypes and the arf2-101, plt1-4, plt2-2 (WS), arf2-101plt1-4and arf2-101plt2-2mutants. Five-day-old seedlings were transferred onto MS medium or MS medium supplementedwith 0, 10 or 30mM ABA for 5 d. The meristem zone is marked with a red line. Bars¼ 50mm. (B) The root meristemzone (MZ) cell number, zone length, and average cell length of each ecotype in (A). Three independent experimentswere conducted with similar results. Each experiment was conducted with 12–25 roots from three plates. Values aremeans� SD. Means with different letters are significantly different at P< 0.01.



regulate PLT2 expression, it is likely that other factorsregulated by ARF2 are involved in mediating thestability of the PLT2 protein. Using a DEX-inducibletransgenic line, 35S:PLT2-GR, we showed that after DEXinduction, cell division was greatly promoted and celldifferentiation was completely blocked in the arf2-101mutant, which did not occur in the WT. This resultsuggests that ARF2 antagonistically functions with PLT2in modulating cell division and differentiation. However,ABA treatment could alleviate the positive role of PLT2 incell division in the arf2-101 mutant, suggesting that ABAsignaling could also inhibit cell division independent ofARF2. Genetic analysis suggests that ARF2 and PLT2 likelywork in the same pathway to regulate root growth.Although PLT2 protein accumulated to a higher level inthe arf2-101 mutant than in the WT, the arf2-101 mutantstill exhibits ABA hypersensitivity in RAM activity,suggesting that PLT1, PIN proteins, and other factorsmust be involved in the ARF2-mediated root growthunder ABA signaling. This also suggests that when PLT2exceeds a certain level, it will promote cell divisionand retard cell differentiation, which is mediated byARF2.

In summary, our study uncovered important roles ofARF2 in coordinating with PLETHORAs and PINs tomediate root meristem activity under ABA signaling inArabidopsis.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana plants of the Columbia ecotype(Col-0), Enkheim-2 ecotype (En) and Wassilewskijaecotype (Ws) were used in this study. The marker linesand mutants used were: ProPLT1-CFP, ProPLT2-CFP,ProPLT1:PLT1-YFP and ProPLT2:PLT2:YFP (Galinha et al.2007), ProDR5-GFP (Benkova et al. 2003), ProPIN1:PIN1-GFP (Benkova et al. 2003), ProPIN4:PIN4-GFP (Vietenet al. 2005), ProPIN3:PIN3-GFP and ProPIN7:PIN7-GFP(Blilou et al. 2005), arf2-101 (Wang et al. 2011), pin 1-1(Okada et al. 1991), pin4-3 (Friml et al. 2004), pin7-2(Benkova et al. 2003), plt1-4, plt2-2 and plt1-4plt2-2(Aida et al. 2004), Pro35S:PLT2-GR (Ding and Friml, 2010)and Pro35S:PIN1 (Benkova et al. 2003).

Seeds were surface-sterilized in 0.5% NaClO solu-tion for 15min, rinsed five times with sterile water,plated on Murashige and Skoog (MS) medium with

2.0% sucrose and 0.9% agar, and then stratified at 4 °Cin the dark for 2 d. Plants were germinated and grownunder long-day conditions (16 h light/8 h dark) at20–22 °C with 60%–70% relative humidity.

For DEX induction analyses, 4-day-old uniformseedlings were transferred to MS medium or MSsupplemented with 30mM ABA, 2mM DEX, or 30mMABA plus 2mM DEX and vertically grown for 3 d beforeanalysis. Alternatively, Arabidopsis seeds were germi-nated and grown on MS medium or MS mediumcontaining 0.3mM ABA, 2mM DEX, or 0.3mM ABA plus2mM DEX for 6 d before analyzing.

The primary root length (mm) and relative rootgrowth (%) were measured from photographs of plates,using the Image J program (Image J; National Institutesof Health; http://rsb.info.nih.gov/ij). Twenty-five rootswere measured from two Petri dishes for eachtreatment and three independent replicates wereconducted for each condition.

Microscopy and confocal microscopyFour- or 5-day-old uniform seedlingswere transferred toMS medium supplemented with different concentra-tions of ABA, or DEX. After 3 or 5 d, the roots werephotographed with an Olympus BX53 microscope. Themeristem cell number was measured as the number ofmeristematic cortex cells from the quiescent center(QC) to the first elongated cell. The root meristem celllength was assessed as the distance between the QCand the first elongating cell (Perilli and Sabatini, 2010).The elongation zone (EZ) cell length and cell numberwas assessed as the distance from the first elongatingcell to the first cell with a root hair.

For confocal microscopy images, 4-day-old uniformseedlings were transferred to MS medium or MSmedium with 0, 10 or 30mM ABA grown for 2 d. Theseedlings were mounted in 10mM propidium iodide for3min before being imaged with a Carl Zeiss LSM510META confocal microscope. The relative fluorescence(%) was measured using Image J.

ACKNOWLEDGMENTS

This work was supported by grants from NationalBasic Research Program of China (973 program,2012CB114300) andNational Science Foundation of China(31421062) to Z. G.

40 Promchuea et al.


http://rsb.info.nih.gov/ij)

AUTHOR CONTRIBUTIONS

Z. G. and S.P. designed the experiments. S. P. performedmost of experiments and analyzed the data. Otherauthors assisted in experiments and discussed theresults. Z. G. and S. P. wrote the manuscript.

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SUPPORTING INFORMATION

Additional Supporting Information may be found onlinein the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.12506/suppinfoFigure S1. The pin1 arf2-101 double mutant is moreresistant to ABA than the arf2-101 single mutant inprimary root growth(A) The root growth phenotypes of the Col-0 and Enecotypes, and the arf2-101, pin1-1, (En) and arf2-101 pin1-1mutants. Five-day-old seedlings were transferred ontoMS medium or MS medium containing 10 or 30mMABAand grown for 5 d. (B) Primary root length in (A).(C) Relative root growth in (B). Three experiments wereconducted with similar results. Each experiment wasconducted with 8-15 roots from three plates.Figure S2. The arf2-101 pin3-4 double mutant exhibitssimilar ABA sensitivity as the arf2-101 single mutant inroot growth(A) Five-day-old seedlings were transferred onto MSmedium or MS medium containing 10 or 30mM ABA andgrown for 5 d. (B) Primary root length in (A). (C) Relativeroot growth in (B). Three experiments were conductedwith similar results. Each experiment was conductedwith 8-15 roots. Values are means� SD. Means withdifferent letters are significantly different at P < 0.01.(D) Root meristem phenotypes of Col-0 (WT) and thearf2-101, pin3-4, and arf2-101 pin3-4 mutant seedlings in(A). The meristem zone is marked with a red line.Bars¼ 50mm.Figure S3. The arf2-101 pin4-3 double mutant is moreresistant to ABA than the arf2-101 single mutant inprimary root growth(A) The root growth phenotypes of the Col-0 and Enecotypes, and the arf2-101, pin4-3 (EN) and arf2-101 pin4-3mutants. Four-day-old seedlings were transferred ontoMS medium or MS medium containing 10 or 30mMABAand grown for 5 d. (B) The primary root length of theseedlings in (A). (C) The relative root length in (B). Threeexperiments were conducted with similar results andeach experiment was conducted with three replicates,each with 8-15 roots from one plate. Values aremeans� SD. Means with different letters are signifi-cantly different at P< 0.01.

42 Promchuea et al.


http://onlinelibrary.wiley.com/doi/10.1111/jipb.12506/suppinfo

http://onlinelibrary.wiley.com/doi/10.1111/jipb.12506/suppinfo

http://onlinelibrary.wiley.com/doi/10.1111/jipb.12506/suppinfo/







































Figure S4. The arf2-101 pin7-2 double mutant showedsimilar ABA sensitivity as the arf2-101 single mutant inroot growth(A) Four-day-old seedlings were transferred to MSmedium or MS medium containing 10 or 30mM ABAand grown for 5 d. (B) The primary root length ofthe seedlings in (A). (C) The relative root length in(B). Three experiments were conducted with similarresults. Each experiment was conducted with threereplicates, each with 8-15 roots from one plate.Values are means� SD. Means with different lettersare significantly different at P < 0.01. (D). Rootmeristem phenotypes of Col-0 (WT) and the arf2-101,pin7-2, and arf2-101 pin7-2 mutant seedlings in (A).The meristem zone is marked with a red line.Bars¼ 50mm.

Figure S5. Overexpression of PLT2 promotes rootmeristem cell division and inhibits cell differentiationduring seed germination in the arf2-101 mutant(A) Seedling growth phenotypes after ABA and DEXtreatment. SeedsweregerminatedonMSmediumorMSmedium supplemented with 2mM DEX, 0.3mM ABA or0.3mMABAplus 2mMDEX for 6 d. (B)Statistical analysesof root meristem number in (C). The experiment wasconducted with three replicates, each with 12�25 rootsfrom one plate. Values are means� SD. (C) Comparisonof the root meristems of the Pro35S:PLT2-GR/Col-0 (left)and Pro35S:PLT2-GR/arf2-101 (right) transgenic seedlingson MS medium supplemented with 2mM DEX. Celldivision, but no cell differentiation, was observed in theroot meristem of the Pro35S:PLT2-GR/arf2-101 transgenicplants with DEX induction. Scale bars¼ 50mm.



































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